JP2019149979A - SECRETORY IMMUNOGLOBULIN A (sIgA)-BINDING NUCLEIC ACID MOLECULE, sIgA DETECTION SENSOR, sIgA DETECTION REAGENT, AND sIgA ANALYSIS METHOD - Google Patents

Abstract

To provide a novel molecule that can be used for detection of sIgA.SOLUTION: The secretory immunoglobulin A (sIgA)-binding nucleic acid molecule of the present invention is characterized in that it contains a polynucleotide of (a) or (b): (a) a polynucleotide consisting of a base sequence of sequence number 1 and (b) a polynucleotide that consists of a base sequence having at least 80% identity to the base sequence of (a) and binds to the sIgA.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an sIgA-binding nucleic acid molecule, an sIgA detection sensor, an sIgA detection reagent, and an sIgA detection method.

  In recent years, stress has been emphasized because stress can contribute to fatigue and depression. However, there is a problem that it is difficult for a person to determine whether or not the person is in a stress state because the person himself / herself is not aware or is subjective. For this reason, establishment of a method for objectively judging stress is required.

  It is known that humans increase the secretion of sIgA in saliva when they feel stress. Therefore, a method of indirectly evaluating stress by measuring sIgA in the saliva has been attempted. Specifically, an ELISA method using an antibody having sIgA as an antigen has been reported (Non-patent Document 1).

  However, since an antibody is a protein and has a problem in stability, it is difficult to use the antibody for a low-cost and simple test method.

Atsuko Towa, “Nurse's job stressors, burnout and physical health issues: Questionnaire and immunological indicators”, Behavioral Medicine Research, Vol. 10, No. 1, pp. 25-33

  Therefore, an object of the present invention is to provide a new molecule that can be used for detection of sIgA.

The sIgA-binding nucleic acid molecule (hereinafter also referred to as “nucleic acid molecule”) of the present invention is characterized by including any of the following polynucleotides (a) and (b).
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 (b) a polynucleotide comprising a nucleotide sequence having 80% or more identity to the nucleotide sequence of (a) and binding to sIgA SEQ ID NO: 1: GGTAAGCCTCGGACCCTAGATTTG U GG UU CA U CCACC U CG U ACCCA U AAGGGCGGC U A U CA UU C U GCACC U GGGAAA U C

  The sensor for detecting sIgA of the present invention comprises the sIgA-binding nucleic acid molecule of the present invention.

  The sIgA detection reagent of the present invention comprises the sIgA-binding nucleic acid molecule of the present invention.

The method for detecting sIgA of the present invention comprises a step of contacting a sample with a nucleic acid molecule and detecting sIgA in the sample,
The nucleic acid molecule is the sIgA-binding nucleic acid molecule of the present invention;
In the detection step, sIgA in the sample is bound to the nucleic acid molecule, and sIgA in the sample is detected by the binding.

  The sIgA-binding nucleic acid molecule of the present invention can bind to sIgA. Therefore, according to the sIgA-binding nucleic acid molecule of the present invention, for example, sIgA can be detected with excellent accuracy depending on the presence or absence of binding to sIgA in a sample.

FIG. 1 is a graph showing the binding ability of aptamers to sIgA in Examples. FIG. 2 is a graph showing the relative value (Relative Unit) of the amount of aptamer binding to sIgA in Examples.

(1) sIgA-binding nucleic acid molecule The sIgA-binding nucleic acid molecule of the present invention is characterized by comprising any of the following polynucleotides (a) or (b).
(A) a polynucleotide comprising the base sequence of SEQ ID NO: 1 (b) a polynucleotide comprising a base sequence having 80% or more identity to the base sequence of (a) and binding to sIgA

  The nucleic acid molecule of the present invention can bind to sIgA as described above. The nucleic acid molecule of the present invention may be bound to, for example, the immunoglobulin heavy chain constituting sIgA, the immunoglobulin light chain, or both. You may couple | bond with the bridge | crosslinking part (J chain) of IgA, and may couple | bond with the secretory component (secretory component) couple | bonded with sIgA. The sIgA is not particularly limited, and examples of its origin include humans and non-human animals. Examples of the non-human animal include mouse, rat, monkey, rabbit, dog, cat, horse, cow, pig and the like. Information on the amino acid sequence of the C region (Ig alpha-1 chain C region) of human Igα-1 chain is registered in, for example, UniProt (http://www.uniprot.org/) accession number P01876, and Information on the amino acid sequence of the C region (Ig alpha-2 chain C region) of human Ig-α2 chain is registered in, for example, UniProt (http://www.uniprot.org/) accession number P01877.

  In the present invention, “binding to sIgA” means, for example, having binding ability to sIgA or having binding activity to sIgA. The binding between the nucleic acid molecule of the present invention and the sIgA can be determined by, for example, surface plasmon resonance molecular interaction (SPR) analysis. For example, ProteON (trade name, BioRad) can be used for the analysis. Since the nucleic acid molecule of the present invention binds to sIgA, it can be used, for example, for detection of sIgA.

  The nucleic acid molecule of the present invention may be, for example, a molecule composed of the polynucleotide (a) or (b) or a molecule containing the polynucleotide. Moreover, the nucleic acid molecule of the present invention may be composed of, for example, DNA, RNA, or DNA and RNA. When the nucleic acid molecule of the present invention is composed of DNA, the nucleic acid molecule of the present invention can be referred to as, for example, a DNA molecule or a DNA aptamer.

  The polynucleotide (a) may be, for example, a polynucleotide comprising the base sequence of SEQ ID NO: 1 or a polynucleotide comprising the base sequence of SEQ ID NO: 1. The polynucleotide (a) may be a nucleotide containing a partial sequence of the base sequence of SEQ ID NO: 1 or a polynucleotide comprising the partial sequence. The partial sequence is not particularly limited. For example, in the base sequence of SEQ ID NO: 1, it may be a sequence in which at least one of the 5 ′ terminal and the 3 ′ terminal is deleted, or the intermediate region is deleted. It may be an array. The polynucleotide of SEQ ID NO: 1 is shown below.

sIgA binding nucleic acid molecule 1 (SEQ ID NO: 1)
GGTAAGCCTCGGACCCTAGATTTG U GG UU CA U CCACC U CG U ACCCA U AAGGGCGGC U A U CA UU C U GCACC U GGGAAA U C

  In (b), “identity” is not particularly limited, and may be any range as long as the polynucleotide of (b) binds to sIgA. The identity is, for example, 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, 96% or more, 97% or more, particularly preferably 98% or more, and most preferably 99%. % Or more. The identity can be calculated with default parameters using analysis software such as BLAST and FASTA (hereinafter the same).

The polynucleotide in the nucleic acid molecule of the present invention may be, for example, the polynucleotide (c) below. In this case, the nucleic acid molecule of the present invention may be, for example, a molecule composed of the following polynucleotide (c) or a molecule containing the following polynucleotide (c).
(C) a polynucleotide comprising a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to the polynucleotide comprising the base sequence of (a) and binding to sIgA

In the above (c), the “hybridizable polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (a) and has a range that binds to the sIgA. That's fine. The hybridization can be detected by, for example, various hybridization assays. The hybridization assay is not particularly limited, for example, Zanburuku (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) ," [Cold Spring Harbor Laboratory Press (1989)] and the like can also be employed.

In the above (c), the “stringent conditions” may be, for example, any of low stringent conditions, medium stringent conditions, and high stringent conditions. “Low stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 32 ° C. “Medium stringent conditions” are, for example, 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 42 ° C. “High stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C. The degree of stringency can be set by those skilled in the art by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, time, and the like. "Stringent conditions" are, for example, Zanburuku previously described (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) ," [Cold Spring Harbor Laboratory Press ( 1989)] etc. may be employed.

The polynucleotide in the nucleic acid molecule of the present invention may be, for example, the polynucleotide (d) below. In this case, the nucleic acid molecule of the present invention may be, for example, a molecule comprising the following polynucleotide (d) or a molecule comprising the following polynucleotide (d).
(D) a polynucleotide consisting of a base sequence in which one or several bases are deleted, substituted, inserted and / or added in the base sequence of (a), and binding to the sIgA

  In the above (d), “one or several” may be, for example, within a range in which the polynucleotide of (d) binds to sIgA. In the base sequence of (a), the “1 or several” is, for example, 1 to 16, 1 to 12, 1 to 10, 1 to 8, 1 to 7, 1 to 5, 1 to 3, 1 to 2, and 1. In the present invention, the numerical range of numbers such as the number of bases and the number of sequences, for example, discloses all positive integers belonging to the range. That is, for example, the description “1 to 5 bases” means all disclosures of “1, 2, 3, 4, 5 bases” (hereinafter the same).

The polynucleotide (b) in the nucleic acid molecule of the present invention may be, for example, the polynucleotide (e) below. In this case, the nucleic acid molecule of the present invention may be, for example, a molecule composed of the following polynucleotide (e) or a molecule containing the following polynucleotide (e).
(E) It consists of a base sequence having 80% or more identity to the base sequence of (a), can form a secondary structure represented by the following formula (2), and binds to sIgA Polynucleotide

In (e), “identity” is not particularly limited, and is the same as (b), for example. In the polynucleotide (e), “can form a secondary structure” means, for example, that the polynucleotide (e) can form the stem structure and the loop structure in the formula (2). . Specifically, “secondary structure can be formed” means that, for example, in the polynucleotide (e), the following bases (e1), (e2) and (e3) form a stem structure, This means that bases other than the bases of e1), (e2), and (e3) form a loop structure. In the polynucleotide (e), the following bases (e1), (e2), and (e3) may form, for example, a stem structure, and base pairs facing each other in the stem structure can form a hydrogen bond. Any combination of bases may be used. Examples of the combination of bases capable of forming a hydrogen bond include AT, TA, AU, UA, GC, and CG.
(E1) A base corresponding to the 5th to 8th bases in the base sequence of SEQ ID NO: 1 and a base corresponding to the 54th to 57th bases (e2) Corresponding to the 14th to 17th bases of the base sequence of SEQ ID NO: 1 A base corresponding to the 49th to 52nd bases (e3) a base corresponding to the 24th to 27th bases of the base sequence of SEQ ID NO: 1 and a base corresponding to the 33rd to 36th bases

The polynucleotide (d) in the nucleic acid molecule of the present invention may be, for example, the following polynucleotide (f). In this case, the nucleic acid molecule of the present invention may be, for example, a molecule composed of the following polynucleotide (f) or a molecule containing the following polynucleotide (f).
(F) The base sequence of (a) comprises a base sequence in which one or several bases are deleted, substituted, inserted and / or added, and forms the secondary structure represented by the formula (2). A polynucleotide capable of binding to said sIgA

  In (f), “one or several” is not particularly limited, and is the same as (d), for example. In the polynucleotide (f), “can form a secondary structure” means, for example, that the polynucleotide (f) can form a stem structure and a loop structure in the above formula. The description of the polynucleotide (e) can be incorporated. The stem structure and loop structure will be described later.

  In the nucleic acid molecule of the present invention, the structural unit of the polynucleotide is, for example, a nucleotide residue, and examples thereof include a deoxyribonucleotide residue and a ribonucleotide residue. As will be described later, the polynucleotide is, for example, DNA composed of deoxyribonucleotide residues, DNA containing deoxyribonucleotide residues and ribonucleotide residues, and may further contain non-nucleotide residues. The nucleic acid molecule of the present invention is hereinafter also referred to as an aptamer, for example.

  The nucleic acid molecule of the present invention may be, for example, a molecule composed of any one of the polynucleotides (a) to (f) or a molecule containing any one of the polynucleotides (a) to (f). In the latter case, the nucleic acid molecule of the present invention may contain two or more of any of the polynucleotides (a) to (f) as described later. The two or more polynucleotides (a) to (f) may have the same sequence or different sequences. In the latter case, the nucleic acid molecule of the present invention may further have, for example, a linker and / or an additional sequence. Here, the linker is, for example, a sequence between polynucleotides, and the additional sequence is, for example, a sequence added to the end.

  For example, when the nucleic acid molecule of the present invention includes a plurality of polynucleotides, it is preferable that the sequences of the plurality of polynucleotides are linked to form a single-stranded polynucleotide. For example, the sequences of the plurality of polynucleotides may be directly linked to each other or indirectly linked via a linker. The polynucleotide sequences are preferably linked directly or indirectly at the respective ends. When a plurality of sequences of the polynucleotide are included, the number of the sequences is not particularly limited, and is 2 or more, 2 to 20, 2 to 10, 2 or 3, for example.

  The length in particular of the said linker is not restrict | limited, For example, it is 1-200 base length, 1-24 base length, 1-20 base length, 3-12 base length, 5-9 base length. The structural unit of the linker is, for example, a nucleotide residue, and examples thereof include a deoxyribonucleotide residue and a ribonucleotide residue. The linker is not particularly limited, and examples thereof include polynucleotides such as DNA consisting of deoxyribonucleotide residues and DNA containing ribonucleotide residues. Specific examples of the linker include polydeoxythymine (poly dT), polydeoxyadenine (poly dA), poly dAdT which is a repeating sequence of A and T, and preferably poly dT and poly dAdT.

  In the nucleic acid molecule of the present invention, the polynucleotide is preferably a single-stranded polynucleotide. The single-stranded polynucleotide is preferably capable of forming a stem structure and a loop structure by, for example, self-annealing. The polynucleotide is preferably capable of forming a stem loop structure, an internal loop structure, and / or a bulge structure, for example.

  The nucleic acid molecule of the present invention may be, for example, double stranded. In the case of a double strand, for example, one single-stranded polynucleotide includes any of the polynucleotides (a) to (f), and the other single-stranded polynucleotide is not limited. Examples of the other single-stranded polynucleotide include a polynucleotide comprising a base sequence complementary to any one of the polynucleotides (a) to (f). When the nucleic acid molecule of the present invention is double-stranded, it is preferably dissociated into a single-stranded polynucleotide by denaturation or the like prior to use. The dissociated single-stranded polynucleotide of any one of (a) to (f) preferably has, for example, a stem structure and a loop structure as described above.

  The structural unit of the nucleic acid molecule of the present invention is, for example, a nucleotide residue. The length of the nucleic acid molecule is not particularly limited. The lower limit of the length of the nucleic acid molecule is, for example, 15 base length, 35 base length, 55 base length, 75 base length. The upper limit of the length of the nucleic acid molecule is, for example, 1000 bases, 200 bases, 100 bases, 90 bases, 80 bases. The range of the length of the nucleic acid molecule is, for example, 15 to 1000 bases, 35 to 200 bases, 55 to 90 bases, 75 to 80 bases.

  Examples of the nucleotide residue include deoxyribonucleotide residue and ribonucleotide residue. Examples of the nucleic acid molecule of the present invention include DNA composed only of deoxyribonucleotide residues, DNA containing one or several ribonucleotide residues, and the like. In the latter case, “one or several” is not particularly limited. For example, in the polynucleotide, for example, 1 to 91, 1 to 30, 1 to 15, 1 to 7, 1 to 3 are used. One or two.

  The polynucleotide may include a natural base or a modified base as a base in a nucleotide residue. The natural base (non-artificial base) is not particularly limited, and examples thereof include a purine base having a purine skeleton and a pyrimidine base having a pyrimidine skeleton. The purine base is not particularly limited, and examples thereof include adenine (a) and guanine (g). The pyrimidine base is not particularly limited, and examples thereof include cytosine (c), thymine (t), uracil (u) and the like.

  When the polynucleotide has the modified base, the site and number are not particularly limited. When the nucleic acid molecule of the present invention has the modified base, in the polynucleotide of SEQ ID NO: 1, for example, part or all of the underlined uracil is a modified base. When the underlined uracil is a modified base, the modified base is preferably a modified uracil modified with a uracil base.

  The modified base is, for example, a base modified with a modifying group. The base modified by the modifying group (modified base) is, for example, the natural base. Examples of the natural base include purine base and pyrimidine base. The modified base is not particularly limited, and examples thereof include modified adenine, modified guanine, modified cytosine, modified thymine, and modified uracil.

  In the modified base, for example, the modified base may be directly modified with the modifying group, or the modified base may be indirectly modified with the modifying group. In the latter case, for example, the modified base is modified with the modifying group via a linker. The linker is not particularly limited.

The modification site of the base to be modified by the modifying group is not particularly limited. When the base is a pyrimidine base, examples of the modification site of the pyrimidine base include the 5th and 6th positions of the pyrimidine skeleton, and the 5th position is preferable. When the 5-position of the pyrimidine base is modified, thymine has a methyl group at the carbon at the 5-position. For example, the modifying group may be bonded directly or indirectly to the carbon at the 5-position. The modifying group may be bonded directly or indirectly to the carbon of the methyl group bonded to the carbon at the 5-position. In the pyrimidine skeleton, when “═O” is bonded to the carbon at the 4-position and a group other than “—CH ₃ ” or “—H” is bonded to the carbon at the 5-position, this is called modified thymine or modified uracil. Can do.

  When the modified base is modified uracil, the modifying group is preferably an adenine residue. That is, in the modified base, for example, the base is modified with the adenine residue. The site where the adenine residue modifies the base to be modified is not particularly limited, and examples thereof include an amino group that binds to the 6-position carbon in the adenine residue. The modified base to be modified with the adenine residue is not particularly limited, but for example, uracil is preferable, and the carbon at the 5-position of uracil is preferably modified with the adenine residue. A part of the modifying group may be further substituted or modified. When the modifying group includes an adenine residue, it is preferable that the hydrogen bonded to the 9th-position nitrogen atom is substituted with a substituent represented by the following formula (6) in the adenine residue. In the following formula (6), n is a positive integer of 1 to 12, preferably 4.

When the modifying group is the adenine residue, for example, it is preferable that the modified base is modified with the modifying group via the linker as shown below.
[Nucleotide residue]-[linker]-[adenine residue]

The linker is not particularly limited. For example, the linker is represented by a formula between the nucleotide residue and the adenine residue as follows, but is not limited thereto. In the following formula, the numerical value of _n in (CH ₂ ) _n is, for example, 1 to 10, _{2 to 10, or 2} .
[Nucleotide residue] -C = CC (= O) -NH- (CH ₂ ) _n- [Adenine residue]
[Nucleotide residue] -C = CC (= O) -NH-CH 2 -CH 2 - [ adenine residue]

In the above formula, one end [-C] of the linker forms, for example, a single bond with the carbon of the base to be modified in the nucleotide residue, and the other end [CH _2- ] of the linker has, for example, an adenine residue. It is bonded to an amine (—NH) (for example, an amine bonded to the 6-position carbon).

The modified uracil base modified with the adenine residue is preferably a modified uracil base represented by the following formula (1).

In the modified uracil of the formula (1), for example, it can also be said that the carbon at the 5-position of uracil is modified with a modifying group of the following formula (3). That is, in the modified uracil of the formula (1), for example, a hydrogen atom bonded to the carbon at the 5-position of uracil is substituted with a modifying group of the following formula (3).

Specific examples of the uridine nucleotide residue modified with the adenine residue in the polynucleotide include a nucleotide residue represented by the following formula (4) (hereinafter also referred to as “KK9”).

  In the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, for example, the underlined uridine nucleotide residue is more preferably KK9.

When the nucleic acid molecule of the present invention has, for example, KK9, for synthesis of the polynucleotide, for example, a nucleotide triphosphate represented by the following formula (5) (hereinafter also referred to as “KK9 monomer”) is used as a monomer. Can be used as a molecule. In the synthesis of the polynucleotide, for example, the monomer molecule binds to another nucleotide triphosphate through a phosphodiester bond. The production method of the KK9 monomer can be produced by a known method. For example, International Publication No. 2015/064223 can be referred to.

  Examples of the modifying group include a methyl group, a fluoro group, an amino group, a thio group, a benzylaminocarbonyl group, a tryptaminocarbonyl group, and an isobutylaminocarbonyl group. It is done.

  Specific examples of the modified adenine include, for example, 7′-deazaadenine. Specific examples of the modified guanine include, for example, 7′-deazaguanine. Specific examples of the modified cytosine include, for example, 5′-methylcytosine (5-Me-dC) and the like. Specific examples of the modified uracil include 5′-benzylaminocarbonyluracil, 5′-tryptaminocarbonyluracil, 5′-isobutylamino. Examples of the modified uracil include 5′-benzylaminocarbonyluracil (BndU), 5′-tryptaminocarbonyluracil (TrpdU), 5′-isobutylaminocarbonyluracil and the like. It is done. The exemplified modified uracil can also be referred to as a modified base of uracil.

  For example, the polynucleotide may include only one of the modified bases, or may include two or more types of the modified bases.

  The nucleic acid molecules of the present invention may include, for example, modified nucleotides. The modified nucleotide may be a nucleotide having the modified base described above, a nucleotide having a modified sugar in which a sugar residue is modified, or a nucleotide having the modified base and the modified sugar.

  The sugar residue is not particularly limited, and examples thereof include deoxyribose residue or ribose residue. The modification site in the sugar residue is not particularly limited, and examples thereof include the 2'-position and the 4'-position of the sugar residue, and both of them may be modified. Examples of the modifying group of the modified sugar include a methyl group, a fluoro group, an amino group, and a thio group.

  In the modified nucleotide residue, when the base is a pyrimidine base, for example, the 2'-position and / or the 4'-position of the sugar residue is preferably modified. Specific examples of the modified nucleotide residue include, for example, a 2′-methylated-uracil nucleotide residue and a 2′-methylated-cytosine nucleotide residue in which the deoxyribose residue or the 2 ′ position of the ribose residue is modified. 2'-fluorinated-uracil nucleotide residues, 2'-fluorinated-cytosine nucleotide residues, 2'-aminated-uracil nucleotide residues, 2'-aminated-cytosine nucleotide residues, 2'-thiolated -Uracyl nucleotide residue, 2'-thiolated-cytosine nucleotide residue and the like.

  The number of the modified base is not particularly limited. In the polynucleotide, the number of the modified base is, for example, one or more. In the polynucleotide, the modified base is, for example, 1 to 80, 1 to 70, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, and All the bases may be the modified bases. The number of the modified bases may be, for example, the number of any one of the modified bases or the total number of the two or more modified bases. Further, the modified base in the full length of the nucleic acid molecule containing the polynucleotide is not particularly limited, and is, for example, 1 to 80, 1 to 50, or 1 to 20, preferably the same as the above-mentioned range. It is.

  In the polynucleotide, the ratio of the modified base is not particularly limited. The ratio of the modified base is, for example, 1/100 or more, 1/40 or more, 1/20 or more, 1/10 or more, 1/4 or more, or 1/3 or more of the total number of bases of the polynucleotide. . Further, the ratio of the modified base in the entire length of the nucleic acid molecule containing the polynucleotide is not particularly limited, and is the same as the above range. Here, the total number of bases is, for example, the total number of natural bases and modified bases in the polynucleotide. The ratio of the modified base is expressed as a fraction, and the total number of bases and the number of modified bases that satisfy this are positive integers.

  When the modified base in the polynucleotide is the modified uracil, the number of the modified uracil is not particularly limited. In the polynucleotide, for example, natural uracil can be replaced with the modified uracil. In the polynucleotide, the number of the modified uracil is, for example, one or more. The modified uracil is, for example, 1 to 80, 1 to 70, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10 in the polynucleotide. All uracils may be the modified uracils.

  In the polynucleotide, the ratio of the modified uracil is not particularly limited. The ratio of the modified uracil is, for example, 1/100 or more, 1/40 or more, 1/20 or more, 1/10 or more, 1/4 or more of the total number of the natural uracil and the modified uracil. 1/3 or more.

  The nucleic acid molecule of the present invention may contain, for example, one or several artificial nucleic acid monomer residues. The “one or several” is not particularly limited, and is, for example, 1 to 100, 1 to 50, 1 to 30, or 1 to 10 in the polynucleotide. Examples of the artificial nucleic acid monomer residue include PNA (peptide nucleic acid), LNA (Locked Nucleic Acid), ENA (2'-O, 4'-C-Ethylene-bridged Nucleic Acids) and the like. The nucleic acid in the monomer residue is the same as described above, for example.

  The nucleic acid molecule of the present invention is preferably resistant to nucleases, for example. The nucleic acid molecule of the present invention preferably has, for example, the modified nucleotide residue and / or the artificial nucleic acid monomer residue for nuclease resistance. Since the nucleic acid molecule of the present invention is nuclease resistant, for example, tens of kDa PEG (polyethylene glycol) or deoxythymidine may be bound to the 5 'end or 3' end.

  The nucleic acid molecule of the present invention may further have an additional sequence, for example. The additional sequence is preferably bound to, for example, at least one of the 5 'end and the 3' end of the nucleic acid molecule, and more preferably the 3 'end. The additional sequence is not particularly limited. The length of the additional sequence is not particularly limited, and is, for example, 1 to 200 bases, 1 to 50 bases, 1 to 25 bases, or 18 to 24 bases. The structural unit of the additional sequence is, for example, a nucleotide residue, and examples thereof include a deoxyribonucleotide residue and a ribonucleotide residue. The additional sequence is not particularly limited, and examples thereof include polynucleotides such as DNA consisting of deoxyribonucleotide residues and DNA containing ribonucleotide residues. Specific examples of the additional sequence include poly dT and poly dA.

  The nucleic acid molecule of the present invention can be used, for example, immobilized on a carrier. In the nucleic acid molecule of the present invention, for example, either the 5 'end or the 3' end is preferably immobilized, and more preferably the 3 'end. When immobilizing the nucleic acid molecule of the present invention, for example, the nucleic acid molecule may be immobilized directly or indirectly on the carrier. In the latter case, for example, it is preferable to immobilize via the additional sequence.

  For example, the nucleic acid molecule of the present invention may further have a labeling substance. Specifically, the labeling substance may be bound to the nucleic acid molecule. The nucleic acid molecule to which the labeling substance is bound can also be referred to as a nucleic acid sensor of the present invention, for example. The labeling substance may be bound to, for example, at least one of the 5 'end and the 3' end of the nucleic acid molecule. The labeling with the labeling substance may be, for example, binding or chemical modification. The labeling substance is not particularly limited, and examples thereof include enzymes, fluorescent substances, dyes, isotopes, drugs, toxins, and antibiotics. Examples of the enzyme include luciferase and NanoLuc luciferase. Examples of the fluorescent substance include pyrene, TAMRA, fluorescein, Cy3 dye, Cy5 dye, FAM dye, rhodamine dye, Texas red dye, JOE, MAX, HEX, TYE and the like, and the dye includes, for example, And Alexa dyes such as Alexa 488 and Alexa 647. The labeling substance may be linked directly to the nucleic acid molecule or indirectly via a linker, for example. The linker is not particularly limited and is, for example, a polynucleotide linker.

  The method for producing the nucleic acid molecule of the present invention is not particularly limited, and can be synthesized by, for example, a genetic engineering technique such as a nucleic acid synthesis method using chemical synthesis or a known method.

  As described above, the nucleic acid molecule of the present invention exhibits binding property to the sIgA. For this reason, the use of the nucleic acid molecule of the present invention is not particularly limited as long as it uses the binding property to sIgA. The nucleic acid molecule of the present invention can be used in various methods, for example, instead of the antibody against sIgA.

  According to the nucleic acid molecule of the present invention, sIgA can be detected. The method for detecting sIgA is not particularly limited, and can be performed, for example, by detecting the binding between sIgA and the nucleic acid molecule with reference to the detection method described later.

(2) Sensor for detecting sIgA As described above, the sensor for detecting sIgA is a sensor for detecting sIgA, and includes the nucleic acid molecule of the present invention. The detection sensor of the present invention only needs to contain the nucleic acid molecule of the present invention, and other configurations are not particularly limited. By using the detection sensor of the present invention, for example, the sIgA can be detected as described above by binding the nucleic acid molecule and the sIgA.

  The detection sensor of the present invention further includes, for example, a carrier, and the nucleic acid molecule is arranged on the carrier. The nucleic acid molecule is preferably immobilized on the carrier. The immobilization of the nucleic acid molecule on the carrier is, for example, as described above. The method of using the detection sensor of the present invention is not particularly limited, and the nucleic acid molecule of the present invention and the detection method of the present invention can be used.

(3) Detection reagent and kit The detection reagent of the present invention comprises the sIgA-binding nucleic acid molecule of the present invention. The detection reagent of this invention should just contain the nucleic acid molecule of the said this invention, and another structure is not restrict | limited at all. When the detection reagent of the present invention is used, for example, the detection of the sIgA can be performed as described above.

  The detection reagent of the present invention may include, for example, the sensor of the present invention as the nucleic acid molecule of the present invention. The detection reagent of the present invention may further have a labeling substance, for example, and the labeling substance may be bound to the nucleic acid molecule. For the labeling substance, for example, the description in the nucleic acid molecule of the present invention can be used. The detection reagent of the present invention may have, for example, a carrier, and the nucleic acid molecule may be immobilized on the carrier. For the carrier, for example, the description in the nucleic acid molecule of the present invention can be incorporated.

  The detection reagent of the present invention may contain other components in addition to the nucleic acid molecule of the present invention, for example. Examples of the component include the carrier, buffer solution, and instruction manual.

  In the detection reagent of the present invention, for example, the other components such as the nucleic acid molecule and the buffer solution may be stored in separate containers, or may be stored in the same container in a mixed or unmixed manner. When the nucleic acid molecule and the other components are contained in separate containers, the detection reagent of the present invention can also be referred to as a detection kit.

(4) Detection method As described above, the detection method of the present invention comprises a step of contacting a sample with a nucleic acid molecule and detecting sIgA in the sample, wherein the nucleic acid molecule is the sIgA-binding nucleic acid of the present invention. In the detection step, sIgA in the sample is bound to the nucleic acid molecule, and sIgA in the sample is detected by the binding. The detection method of the present invention is characterized by using the nucleic acid molecule of the present invention, and other steps and conditions are not particularly limited. In the detection method of the present invention, the sIgA detection sensor of the present invention may be used as the nucleic acid molecule of the present invention.

  According to the present invention, since the nucleic acid molecule of the present invention specifically binds to sIgA, for example, sIgA in a sample can be specifically detected by detecting the binding between sIgA and the nucleic acid molecule. It is. Specifically, since the detection method of the present invention can detect, for example, the presence or absence of sIgA or the amount of sIgA in a sample, it can be said that qualitative or quantitative determination is possible. The detection method of the present invention can also be referred to as an analysis method because, for example, qualitative analysis or quantitative analysis is possible.

  In the present invention, the sample is not particularly limited. Examples of the sample include saliva, urine, plasma and serum.

  The sample may be, for example, a liquid sample or a solid sample. For example, the sample is preferably a liquid sample because it is easy to contact with the nucleic acid molecule and is easy to handle. In the case of the solid sample, for example, a mixed solution, an extract, a dissolved solution, and the like may be prepared using a solvent and used. The solvent is not particularly limited, and examples thereof include water, physiological saline, and buffer solution.

  The detection step includes, for example, a contact step in which the sample and the nucleic acid molecule are brought into contact to bind sIgA in the sample and the nucleic acid molecule, and binding detection in which binding between the sIgA and the nucleic acid molecule is detected. Process. The detection step further includes, for example, a step of detecting the presence or absence or amount of sIgA in the sample based on the result of the binding detection step.

  In the contacting step, the method for contacting the sample and the nucleic acid molecule is not particularly limited. The contact between the sample and the nucleic acid molecule is preferably performed in a liquid, for example. The liquid is not particularly limited, and examples thereof include water, physiological saline, and buffer solution.

  In the contacting step, the contact condition between the sample and the nucleic acid molecule is not particularly limited. The contact temperature is, for example, 4 to 37 ° C. and 18 to 25 ° C., and the contact time is, for example, 10 to 120 minutes and 30 to 60 minutes.

  In the contacting step, the nucleic acid molecule may be, for example, an immobilized nucleic acid molecule immobilized on a carrier or an unimmobilized free nucleic acid molecule. In the latter case, for example, the sample is contacted in a container. The nucleic acid molecule is preferably, for example, the immobilized nucleic acid molecule because of its excellent handleability. The carrier is not particularly limited, and examples thereof include a substrate, a bead, and a container. Examples of the container include a microplate and a tube. The nucleic acid molecule is immobilized as described above, for example.

  As described above, the binding detection step is a step of detecting the binding between sIgA in the sample and the nucleic acid molecule. By detecting the presence or absence of binding between the two, for example, the presence or absence of sIgA in the sample can be detected (qualitative), and by detecting the degree of binding between the two (binding amount), for example, the sample The amount of sIgA in it can be detected (quantified).

  When the binding between the sIgA and the nucleic acid molecule cannot be detected, it can be determined that sIgA is not present in the sample. When the binding is detected, it can be determined that sIgA is present in the sample. .

  A method for detecting the binding between the sIgA and the nucleic acid molecule is not particularly limited. As the method, for example, a conventionally known method for detecting binding between substances can be adopted, and specific examples thereof include the SPR described above. The binding may be, for example, detection of a complex of the sIgA and the nucleic acid molecule.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples. Commercial reagents were used based on those protocols unless otherwise indicated.

[Example 1]
Regarding the sIgA nucleic acid molecule of the present invention, the binding ability and kinetic parameters for sIgA were confirmed by SPR.

(1) Aptamer The sIgA-binding nucleic acid molecule 1 that was synthesized from the polynucleotide (sIgA-binding nucleic acid molecule 1) having the base sequence of SEQ ID NO: 1 and used as the DNA aptamer of the example was the underlined portion in the base sequence of SEQ ID NO: 1. The uridine nucleotide residue was designated as KK9 represented by the above formula (4).

  The sIgA-binding nucleic acid molecule 1 was added with polydeoxyadenine (poly dA) having a length of 20 bases at the 3 'end, and used as a poly dA-added aptamer in SPR described later.

(2) Sample Commercially available human sIgA (IgA (Secretory), Human, MP Biomedicals, LLC-Cappel Products, catalog number: # 55905) was used as a sample in the following tests.

(3) Analysis of binding ability by SPR For analysis of binding ability, ProteON XPR36 (BioRad) was used according to the instruction manual.

  First, as a sensor chip exclusively for ProteON, a chip (trade name: ProteOn NLC Sensor Chip, BioRad) on which streptavidin was immobilized was set in ProteON XPR36. 5 μmol / L of biotinylated poly dT was injected into the flow cell of the sensor chip using ultrapure water (DDW) and allowed to bind until the signal intensity (RU: Resonance Unit) was about 900 RU. The biotinylated poly dT was prepared by biotinylating the 5 'end of 20 base long deoxythymidine. Then, the binding nucleic acid molecule 1 added with 200 nmol / L of the poly dA is injected into the flow cell of the chip for 80 seconds at a flow rate of 25 μL / min using an SPR buffer, and the binding is performed until the signal intensity reaches about 800 RU. I let you. This result is referred to as an aptamer solidification measurement value (A) as a signal indicating the amount of solidification of the aptamer to the sensor chip. Subsequently, the sample was injected with an SPR buffer at a flow rate of 50 μL / min for 120 seconds, and subsequently washed with the SPR buffer flowing for 300 seconds under the same conditions. The human sIgA concentration in the sample was 400 nmol / L. In parallel with the injection of the sample and the washing with the SPR buffer, the signal intensity was measured. The average value of the signal intensity between 115 and 125 seconds is determined with the injection start as 0 seconds, and this is referred to as a protein binding measurement value (B) as a signal indicating the amount of binding between the aptamer and the protein. Then, a value (B / A) obtained by dividing the protein binding measurement value (B) by the aptamer solidification measurement value (A) was obtained as a relative value (Relative Unit). In addition, the control 1 measured the signal intensity in the same manner except that instead of the sIgA-binding nucleic acid molecule 1, a control nucleic acid molecule (SEQ ID NO: 2) consisting of the base sequence of SEQ ID NO: 2 was used, Relative values were obtained. In the base sequence of SEQ ID NO: 2 below, N represents A, U, G, or C, and U in N and U in the underlined part were KK9 represented by the above formula (4). Further, as a negative control, signal intensity was measured in the same manner except that a sample containing human α-amylase (α-Amylase, manufactured by Lee Biosolutions, catalog number: # 120-10) was used instead of human sIgA. The relative value was obtained. These results are shown in FIGS.

Control nucleic acid molecule (SEQ ID NO: 2)
5'-GGTAAGCCTCGGACCCTAGATTTGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGC U A U CA UU C U GCACC U GGGAAA T C-3 '

The composition of the SPR buffer is 40 mmol / L HEPES, 125 mmol / L NaCl, 1 mmol / L MgCl ₂ , 5 mmol / L KCl, and 0.01% Tween (registered trademark) 20, and the pH is 7.4. did.

  FIG. 1 is a graph showing measured values of signal intensity. 1A is a graph showing the binding ability of sIgA-binding nucleic acid molecule 1 to sIgA or α-amylase, and FIG. 1B is a graph showing the binding ability of a control nucleic acid molecule to sIgA or α-amylase. It is. 1 (A) and 1 (B), the horizontal axis indicates the elapsed time with reference to the start of sample injection, and the vertical axis indicates the relative value (RU) of the binding force. As shown in FIGS. 1 (A) and 1 (B), sIgA-binding nucleic acid molecule 1 bound to sIgA, but the control nucleic acid molecule did not bind to sIgA. In addition, as shown in FIGS. 1A and 1B, the sIgA-binding nucleic acid molecule 1 and the control nucleic acid molecule did not bind to α-amylase.

  Next, the result of the relative value (B / A value) is shown in FIG. FIG. 2 is a graph showing the relative values of the binding amount of sIgA-binding nucleic acid molecule 1 and the control nucleic acid molecule to sIgA. In FIG. 2, the horizontal axis indicates the type of nucleic acid molecule and sample. From the left, as a result of α-amylase and sIgA when sIgA-binding nucleic acid molecule 1 is used as the nucleic acid molecule, the control nucleic acid molecule is used as the nucleic acid molecule. The results of α-amylase and sIgA are shown. As shown in FIG. 2, the control nucleic acid molecule did not bind to sIgA and α-amylase. In contrast, sIgA-binding nucleic acid molecule 1 did not bind to α-amylase, but bound to sIgA.

  From these results, it was found that the aptamer of the present invention exhibits binding to the human sIgA.

  As mentioned above, although this invention was demonstrated with reference to embodiment and an Example, this invention is not limited to the said embodiment and Example. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

（付記５）
前記ポリヌクレオチドにおいて、前記配列番号１の塩基配列における下線部のウラシル塩基が、修飾塩基である、付記２から４のいずれか一項に記載のｓＩｇＡ結合核酸分子。
（付記６）
前記（ｂ）のポリヌクレオチドが、下記（ｅ）のポリヌクレオチドである、付記１から５のいずれかに記載の核酸分子。
（ｅ）前記（ａ）の塩基配列に対して、８０％以上の同一性を有する塩基配列からなり、下記式（２）で表される二次構造を形成可能であり、ｓＩｇＡに結合するポリヌクレオチド

（付記７）
前記ポリヌクレオチドが、ＤＮＡである、付記１から６のいずれか一の付記に記載のｓＩｇＡ結合核酸分子。
（付記８）
付記１から７のいずれかに記載の分泌型免疫グロブリンＡ（ｓＩｇＡ）結合核酸分子を含むことを特徴とする、ｓＩｇＡ検出用センサ。
（付記９）
付記１から７のいずれかに記載の分泌型免疫グロブリンＡ（ｓＩｇＡ）結合核酸分子を含むことを特徴とする、ｓＩｇＡ検出試薬。
（付記１０）
試料と核酸分子とを接触させ、前記試料中の分泌型免疫グロブリンＡ（ｓＩｇＡ）を検出する工程を含み、
前記核酸分子が、付記１から７のいずれか一の付記に記載のｓＩｇＡ結合核酸分子であり、
前記検出工程において、前記試料中のｓＩｇＡと前記核酸分子とを結合させて、前記結合により、前記試料中のｓＩｇＡを検出することを特徴とする、ｓＩｇＡの検出方法。
（付記１１）
前記試料が、唾液、尿、血漿、および血清からなる群から選択された少なくとも１つである、付記１０記載のｓＩｇＡの検出方法。 <Appendix>
Some or all of the above embodiments and examples can be described as the following supplementary notes, but are not limited thereto.
(Appendix 1)
A secretory immunoglobulin A (sIgA) -binding nucleic acid molecule comprising the polynucleotide of any of the following (a) or (b):
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 (b) a polynucleotide comprising a nucleotide sequence having 80% or more identity to the nucleotide sequence of (a) and binding to sIgA SEQ ID NO: 1: GGTAAGCCTCGGACCCTAGATTTG U GG UU CA U CCACC U CG U ACCCA U AAGGGCGGC U A U CA UU C U GCACC U GGGAAA U C
(Appendix 2)
The sIgA-binding nucleic acid molecule according to appendix 1, wherein the binding nucleic acid molecule comprises a modified base whose base is modified with a modifying group.
(Appendix 3)
The sIgA-binding nucleic acid molecule according to appendix 2, wherein the modified base is a modified uracil base obtained by modifying a uracil base with a modifying group.
(Appendix 4)
The sIgA-binding nucleic acid molecule according to appendix 3, wherein the modified uracil base is a modified uracil base represented by the following formula (1).

(Appendix 5)
The sIgA-binding nucleic acid molecule according to any one of appendices 2 to 4, wherein in the polynucleotide, the underlined uracil base in the base sequence of SEQ ID NO: 1 is a modified base.
(Appendix 6)
The nucleic acid molecule according to any one of appendices 1 to 5, wherein the polynucleotide (b) is a polynucleotide (e) below.
(E) a base sequence having 80% or more identity to the base sequence of (a), capable of forming a secondary structure represented by the following formula (2), and binding to sIgA nucleotide

(Appendix 7)
The sIgA-binding nucleic acid molecule according to any one of appendices 1 to 6, wherein the polynucleotide is DNA.
(Appendix 8)
A sensor for detecting sIgA, comprising the secretory immunoglobulin A (sIgA) -binding nucleic acid molecule according to any one of appendices 1 to 7.
(Appendix 9)
A sIgA detection reagent comprising the secretory immunoglobulin A (sIgA) -binding nucleic acid molecule according to any one of appendices 1 to 7.
(Appendix 10)
Contacting the sample with a nucleic acid molecule and detecting secretory immunoglobulin A (sIgA) in the sample,
The nucleic acid molecule is the sIgA-binding nucleic acid molecule according to any one of supplementary notes 1 to 7,
In the detection step, sIgA in the sample is bound to the nucleic acid molecule, and sIgA in the sample is detected by the binding.
(Appendix 11)
The method for detecting sIgA according to appendix 10, wherein the sample is at least one selected from the group consisting of saliva, urine, plasma, and serum.

As described above, the sIgA-binding nucleic acid molecule of the present invention can bind to sIgA. Therefore, according to the sIgA-binding nucleic acid molecule of the present invention, for example, sIgA can be detected with excellent accuracy depending on the presence or absence of binding to sIgA in a sample. Therefore, the sIgA-binding nucleic acid molecule of the present invention can be said to be an extremely useful tool for detecting sIgA in fields such as preventive medicine, health care, diagnosis of infectious diseases, and diagnosis of stress.

Claims (10)

A secretory immunoglobulin A (sIgA) -binding nucleic acid molecule comprising the polynucleotide of any of the following (a) or (b):
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 (b) a polynucleotide comprising a nucleotide sequence having 80% or more identity to the nucleotide sequence of (a) and binding to sIgA SEQ ID NO: 1: GGTAAGCCTCGGACCCTAGATTTG U GG UU CA U CCACC U CG U ACCCA U AAGGGCGGC U A U CA UU C U GCACC U GGGAAA U C The sIgA-binding nucleic acid molecule according to claim 1, wherein the binding nucleic acid molecule comprises a modified base in which the base is modified with a modifying group. The sIgA-binding nucleic acid molecule according to claim 2, wherein the modified base is a modified uracil base obtained by modifying a uracil base with a modifying group. The sIgA-binding nucleic acid molecule according to claim 3, wherein the modified uracil base is a modified uracil base represented by the following formula (1).

The sIgA-binding nucleic acid molecule according to any one of claims 2 to 4, wherein the underlined uracil base in the base sequence of SEQ ID NO: 1 is a modified base in the polynucleotide. The nucleic acid molecule according to any one of claims 1 to 5, wherein the polynucleotide (b) is a polynucleotide (e) below.
(E) a base sequence having 80% or more identity to the base sequence of (a), capable of forming a secondary structure represented by the following formula (2), and binding to sIgA nucleotide

The sIgA-binding nucleic acid molecule according to any one of claims 1 to 6, wherein the polynucleotide is DNA. A sensor for detecting sIgA, comprising the secretory immunoglobulin A (sIgA) -binding nucleic acid molecule according to any one of claims 1 to 7. A sIgA detection reagent comprising the secretory immunoglobulin A (sIgA) -binding nucleic acid molecule according to any one of claims 1 to 7. Contacting the sample with a nucleic acid molecule and detecting secretory immunoglobulin A (sIgA) in the sample,
The nucleic acid molecule is a sIgA-binding nucleic acid molecule according to any one of claims 1 to 7,
In the detection step, sIgA in the sample is bound to the nucleic acid molecule, and sIgA in the sample is detected by the binding.

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